CN111713602A - Preparation method and application of chlamydospore licking block of nematophagous fungus - Google Patents
Preparation method and application of chlamydospore licking block of nematophagous fungus Download PDFInfo
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- CN111713602A CN111713602A CN202010651623.0A CN202010651623A CN111713602A CN 111713602 A CN111713602 A CN 111713602A CN 202010651623 A CN202010651623 A CN 202010651623A CN 111713602 A CN111713602 A CN 111713602A
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Abstract
The invention provides a preparation method of a chlamydospore licking block of nematophagous fungi, which is characterized by comprising the following steps: the licking block component adopted by the preparation method comprises the following components in parts by weight: 5-10 parts of urea, 15-20 parts of molasses, 10-25 parts of salt, 5-10 parts of sodium bentonite, 0-2 parts of carboxymethyl cellulose, 0-5 parts of monocalcium phosphate, 1-1.5 parts of compound vitamin, 20-30 parts of bran and 1-2 parts of peanut oil,D.flagrans1-2 parts of chlamydospore powder, wherein the licking block contains 0.5-2 × 104/g of D.flagrans chlamydospores, and the licking block is used for feeding cattle and sheep and has the insecticidal rate of more than 83% on gastrointestinal nematodes.
Description
Technical Field
The invention relates to the technical field of preparation of licking blocks for feeding domestic ruminants, in particular to a preparation method and application of a chlamydospore licking block of nematophagous fungus.
Background
The gut nematodes of domestic ruminants are a major parasitic disease that occurs worldwide and cause severe economic losses in animal husbandry. The main harm of the disease to cattle and sheep is chronic nutrition consumption, production performance reduction and growth retardation, and severe people die from cachexia finally along with the development of disease conditions. For a long time, such epidemic diseases have relied primarily on chemical drugs to prevent and treat insect repellents. However, with the widespread use of anthelmintics, resistance of current animal endoparasites to the three general classes of anthelmintics (benzimidazoles, avermectins/ivermectins, levamisole) frequently occurs and is prevalent and prevalent in some countries or regions. To prevent the continued spread of parasite resistance, researchers recommend rotation, elimination of pasture feces, use of plants that naturally contain tannin, use of vaccines that harbor antigens, and use of natural enemies of nematodes for biological control; the nematode-eating fungi are used for killing infectious larvae (L3) in the environment to control infection sources, so that the method not only saves manpower, material resources and application cost, but also is one of the most direct and effective control strategies with application prospects at present.
Nematophagous fungiArthrobotrys(Duddingtonia)flagrans(synonyms of the same thingsTrichotheciumflagrans) Has been extensively studied and valued in the biological control of ruminant parasites, and in most of the literature the habit of this bacterium is known asDuddingtoniaflagrans。D. flagransDuring culture, a large number of double-walled chlamydospores are produced, which are resistant to the digestive enzymes in the animal's digestive tract without being inactivated. Therefore, spores of the strain can be added into animal feed and discharged out of the body along with fecal ova after passing through the digestive tract, so that a large number of parasitic nematode larvae hatched in feces can be killed, the quantity of L3 is reduced, and the aim of biological prevention and control is fulfilled.
However, since the domestic lack of excellent candidate strainsD. flagransThis severely limits the research progress of animal parasitic nematode biocontrol, and the introduction of foreign species carries the risk of genetic recombination and species invasion. Therefore, the nematophagous fungi are widely separated and identified at 87 sites in 20 provinces (autonomous region/direct prefecture city) and 48 counties in China since 2011, and are separated and obtained for the first time in ChinaD. flagrans13 strains, and the morphology, molecular genetics, predation mechanism, in vitro insecticidal test, in vivo animal digestive tract test and other biological characteristics of local isolates are comprehensively researched, and the results show that the isolated strains in ChinaD. flagransThe strain is cultured in morphologyThe strain is different from foreign strains in sex, is proved to be a new isolate adapted to the geographical characteristics of China, and has great potential as a candidate strain for developing a biocontrol agent.
To achieve the biological control of parasitic nematodes in livestock, the biological control is not only maintainedD. flagransThe spores have stable activity and can be transferred into animal excrement to effectively kill the L3, and the spores need to be put into feed for feeding, storage and transportation. A good dosage form is the key of commercialization of the biocontrol agent, and has been adopted in ChinaD. flagransThe vacuum freeze-drying preparation of the isolate chlamydospore needs large-scale equipment, has larger power consumption and high product cost, and is very difficult to effectively feed a trace amount of freeze-drying preparation to animals; the chlamydospore survival rate is greatly reduced in this process, the germination rate is very low and is not sufficient to effectively kill L3.
In addition, patent CN201610578955.4 discloses a powdery preparation of nematode-trapping fungi and its application; inoculating nematophagous fungus isolate on culture medium (preferably corn-wheat combined grain culture medium), culturing chlamydospore in batch at 25-28 deg.C and relative humidity above 80%, terminating culture for 15-25 days, and collecting culture containing spores. Collecting culture containing spore and grains, spreading in a container, and air drying at 35 deg.C for 3-5 days; or vacuum drying at 35-40 deg.C under vacuum degree of 0.082-0.085 MPa; or naturally drying, and finally harvesting the dried substance for standby preparation and crushing. And mixing the dried culture with bran according to the mass ratio of 1:5 ‒ 1:10, crushing the mixture by a crusher, and uniformly stirring to obtain the powdery preparation of the nematode-trapping fungi. The obtained subpackaged preparation is culture of fungi. The cultures cannot be stored for a long period of time. The bacterial powder stored for several months has obviously reduced insecticidal rate, and can not effectively kill L3, thereby failing to achieve the purpose of biological prevention and control of gastrointestinal parasitic nematodes.
In summary, the prior art has the following disadvantages: the chlamydospore preparation of nematophagous fungi in the prior art is difficult to be effectively eaten by animals; the chlamydospore preparation of nematophagous fungi in the prior art is not storable, and the insecticidal rate is obviously reduced after the chlamydospore preparation is stored under common indoor conditions.
Disclosure of Invention
O.11210, preservation date 2015, 8 months and 31 days; the preservation unit: china general microbiological culture Collection center (CGMCC), which is abbreviated as CGMCC; and (4) storage address: western road No.1, north chen west city of township, 3, institute for microbiology, china academy of sciences, common microbiology center of the committee for preservation and management of microbial cultures, china, and zip code 100101. The SDH035 isolate was assigned the sequence accession number KP2575930 at NCBI (GenBank).
Chlamydospore production: recovering SDH035 strain preserved on 2% corn flour agar (CMA) slant culture medium, selecting partial hypha or spore with inoculating loop to 0.4% CMA, culturing in 30 deg.C constant temperature incubator for 5-7 d, taking 4 pieces of agar block with length of 4mm × 4mm from colony growth edge, inoculating to 50 mL potato glucose (PD) liquid culture medium, shaking at 180 r/min and 30 deg.C for 6d, enlarging and culturing the liquid cultured seed by PD broth according to 10% inoculum size for 1 time in the same way as above, inoculating to sterilized grain culture medium according to 5-10% seed size of grain culture medium (corn + wheat =2: 1), culturing in 25 deg.C constant temperature incubator for 21d in dark, taking grain with mycelium, adding appropriate amount of 0.01% Tween-80 solution to elute chlamydospore, filtering the bacterial solution with 4 layers of gauze to remove mycelium, counting with a blood cell counting plate, and calculating chlamydospore per ml.
A preparation method of a chlamydospore lick block of nematophagous fungi comprises the following steps:
a preparation method of a chlamydospore licking block of nematophagous fungi is characterized in that: the licking block component adopted by the preparation method comprises the following components in parts by weight: 5-10 parts of urea, 15-20 parts of molasses, 10-25 parts of salt, 5-10 parts of sodium bentonite, 0-2 parts of carboxymethyl cellulose, 0-5 parts of monocalcium phosphate, 1-1.5 parts of compound vitamin, 20-30 parts of bran and 1-2 parts of peanut oil,D. flagrans1-2 parts of chlamydospore powder, wherein the licking block contains 0.5-2 × 104Per g isD. flagransChlamydospores. The lick block prepared: the spore germination rate reaches 27.32-38.65%The above.
The preparation method comprises the following steps: the adopted strain is flagellum type ArthrobotrysArthrobotrysflagransIsolate SDH035 with preservation number CGMCC NO. 11210.
The preparation method comprises the following steps: comprises thatD. flagransPreparation of chlamydospore powder: flagellate type arthrobotrys of nematode-trapping fungiD. flagransAnd carrying out batch culture on the SDH035 isolate on a corn-wheat combined grain culture medium, wherein the culture temperature is 25-30 ℃, the relative humidity is 80-100%, and the culture time is 15-25 d, and then drying the grain containing spores until the moisture content in the grain is below 15%.
The content of spores in the prepared spore powder is 1-2 × 106Per gram.
The preparation method comprises the following steps: the preparation method comprises the following steps: firstly, 30% of bran powder by mass is weighed, then the spore powder is added into the bran powder, and the two are fully and uniformly mixed; the bran powder containing the spore powder is then thoroughly mixed with the remaining bran powder and the remaining components of the formulation.
Further, the licking block component comprises the following components in parts by weight: 25 parts of urea, 75 parts of molasses, 105 parts of NaCl, 25 parts of sodium bentonite, 15 parts of carboxymethyl cellulose, 10 parts of monocalcium phosphate, 5 parts of composite vitamin, 225 parts of bran powder, 5 parts of spore powder and 10 parts of edible peanut oil.
The application of the chlamydospore lick block of the nematophagous fungus is characterized in that: the licking block is used for feeding cattle and sheep, and the insecticidal rate of gastrointestinal nematodes reaches more than 83%.
Compared with the prior art, the invention has the beneficial effects that:
(1) the chlamydospore licking block of the nematophagous fungus prepared by the invention has high spore germination rate, and the spore germination rate in the licking block reaches over 27.32-38.65%.
(2) The biological control method has obvious effect on the biological control of the gastrointestinal nematode diseases of the cattle and sheep; the chlamydospore lick block of the nematophagous fungus prepared by the invention is used for feeding sheep, and the insecticidal rate reaches more than 83 percent.
(3) The chlamydospore lick block of the nematophagous fungus prepared by the invention is storage-resistant and does not depend on a protective agent; under the condition of no addition of a protective agent, the licking block still keeps the insecticidal rate of more than 90 percent when stored for 7 months under the daily indoor condition.
(4) The drug resistance gene of animal parasitic nematodes to anthelmintics is widely spread in the breeding industry all over the world, and has posed a great threat to animal husbandry production. The use of nematophagous fungi for biological control and the like is recommended. Application of nematophagous fungiD.flagransChlamydospore licks can be used to reduce free life stages of parasites in the environment, such as eggs, larvae at stage 1-3, by feeding domestic animals. Compared with vaccine development, the preparation has the advantages of small technical difficulty, low cost and long validity period, and directly controls source infection, so the preparation is a chemical substitution or supplement method with the most application prospect, and is a control method conforming to natural ecological balance. The preparation method is simple, the operation flow is less, complex equipment is not needed, the cost is reduced, and the preparation method is more beneficial to large-scale production, storage and transportation. In addition, the licking block can effectively prevent and control parasitic nematodes of animals while supplementing daily required nutrition for the animals, and the prevention and control strategy belongs to the field of using nematode-eating fungi contained in the licking blockD. flagransAnd the third-stage larvae in the excrement are killed, so that the nematode infection of the animals is reduced from the source. The product of the invention has the characteristics of easy storage, transportation, simple production process, easy large-scale production, convenient production practice and application and low cost.
Drawings
FIG. 1 is a diagram of the appearance of a licking block of chlamydospores of the nematophagous fungus;
FIG. 2 is a graph showing the results of the measurement of spore germination rates of chlamydospore lick blocks in example 4;
FIG. 3 shows the results of in vitro insecticidal effect of chlamydospore lick blocks stored under 6 different conditions for 1-7 months in example 6.
Detailed Description
Example 1 preparation method of chlamydospore licking block of nematophagous fungi
Step 1, separation, purification and identification of strains
9 months in 2013, from sheep manure pile samples from Wuzhou region in Ningxia and sheep manure samples from Yichun city in Heilongjiang provinceThe product is separated by improved plate spreading method, wherein third stage larva of ovine nematode is added as bait into plate, incubated at 25 deg.C, examined once daily or every other day under inverted microscope for 3 weeks, after conidia grow out, single conidia is selected and placed in another single predation structure with 0.2% bran or nematode to be predated, transferred into agar medium containing 0.2% bran, and cultured at 25 deg.C until pure culture is obtained. The strain conidium morphology and data measurements were obtained by slide insertion and strain DNA was extracted and primers ITS1 and ITS4 were used to amplify the 5.8S, ITS-1 and ITS-2 sequences in rDNA. The result is that the SDH035 strain is obtained by separating and purifying 5 parts of sheep manure pile samples, and the morphological and molecular identification shows that the obtained separated strain and the SDH035 strain are obtainedA.flagransThe strain is characterized in that bacterial colonies in 2% corn flour agar are white, hyphae are sparse, hyphae are transparent, separated and branched, conidia can be produced after culture for 2-3 d, a large amount of chlamydospores can be produced after the culture for 7 d, conidia are few, conidia peduncles are transparent, upright and separated, the length of the conidia is 117.5-387.5 (average is 241.59) mu m, the width of the base part is 5-7.5 mu m, the width of the end part is 2.5-5 mu m, the conidia are oval, long oval and elliptical, 1 partition is mainly used, 2 partitions or 3-4 partitions are occasionally seen even if the conidia are 0 partition, the length of the conidia is 20-55.5 (average is 35.89) mu m, the width of the conidia is 10-17 (average is 12.87) mu m, and the strain can produce a large amount of round or elliptical chlamydospores after culture for 3 d at 28 ℃ in a corn and wheat grain combined culture medium (the yield can reach 6-6 × 10)4One per gram) with a chlamydospore diameter of 14-30 (average 25.0) μm.
Step 2, preparation of spore powder
Flagellate type arthrobotrys of nematode-trapping fungiD. flagransAnd (3) carrying out batch culture on the SDH035 isolate on a corn-wheat combined grain culture medium, wherein the culture temperature is 25-30 ℃, the relative humidity is 80-100%, and the culture is stopped for 15-25 d, and then placing the grain containing spores at 38 ℃ for ventilation drying for 48h or placing the grain at room temperature for natural drying until the water content in the grain is kept below 15%. Weighing 60g of the above dried grain culture at random, and detecting sporeThe quantity of the spore reaches 10 by detection6Per gram.
Finally, placing the dried grain culture in a grinder, grinding and packaging the grain culture, and placing the grain culture in a plastic bag for storage under the conditions of dryness and shade; obtaining spore powder with spore content of 106Per gram.
The detection method of the spore number comprises the following steps:
repeatedly eluting chlamydospores in the detected grain culture for 2-3 times by using 0.02% sterile Tween-80, filtering by using 4-6 layers of gauze to obtain chlamydospore suspension, counting by using a blood cell counting plate, and further calculating the content of spores in each gram of dry grains.
Step 3, preparation of lick block
The licking block adopts the following formula: 25 g of urea, 75 g of molasses, 125 g of NaCl, 25 g of sodium bentonite, 5g of carboxymethyl cellulose, 10 g of monocalcium phosphate, 5g of vitamin complex, 225 g of bran powder and 5g of spore powder.
The compound vitamin comprises the following components in percentage by weight: vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, vitamin C and vitamin H, wherein the mass ratio of the vitamin B1 to the vitamin B2 to the vitamin B3 to the vitamin B5 is 1:1: 2: 2.
Firstly weighing 30% of bran powder by mass, then adding the spore powder into the bran powder, and fully mixing the two, then fully mixing the bran powder containing the spore powder, the rest bran powder and the rest components in the formula to ensure that each gram of the material finally contains 1 × 104Per g ofD. flagransThe chlamydospores of the chlamydospores,
the licking block is prepared by pressing the licking block in a brick making machine under the pressure of 18-30 Mpa, and the licking block is obtained after drying at room temperature, the prepared finished product is insoluble in water, and the chlamydospore content is 1 × 104Per gram.
Example 2 preparation method of chlamydospore licking block of nematophagous fungi
The same preparation method as that of example 1, except that the formula adopted by the lick block is changed as follows: 25 g of urea, 75 g of molasses, 115g of NaCl, 25 g of sodium bentonite, 10 g of carboxymethyl cellulose, 10 g of monocalcium phosphate, 5g of compound vitamin, 225 g of bran powder, 5g of spore powder and 5g of edible peanut oil.
The compound vitamin comprises the following components in percentage by weight: vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, vitamin C and vitamin H, wherein the mass ratio of the vitamin B1 to the vitamin B2 to the vitamin B3 to the vitamin B5 is 1:1: 2: 2.
The obtained product is insoluble in water, and has chlamydospore content of 1 × 104Per gram.
Embodiment 3 preparation method of chlamydospore licking block of nematophagous fungi
The same preparation method as that of example 1, except that the formula adopted by the lick block is changed as follows: 25 g of urea, 75 g of molasses, 105g of NaCl, 25 g of sodium bentonite, 15g of carboxymethyl cellulose, 10 g of monocalcium phosphate, 5g of vitamin complex, 225 g of bran powder, 5g of spore powder and 10 g of edible peanut oil.
The compound vitamin comprises the following components in percentage by weight: vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, vitamin C and vitamin H, wherein the mass ratio of the vitamin B1 to the vitamin B2 to the vitamin B3 to the vitamin H is 12: 7: 9: 10: 6:6: 6: 10.
The obtained product is insoluble in water, and has chlamydospore content of 1 × 104Per gram (the shape chart of the licking block is shown in figure 1).
Example 4 spore germination Rate determination of chlamydospore lick blocks
The lick blocks prepared in examples 1-3 were each crushed, sieved through a 80 mesh sieve, diluted to about 600 chlamydospores/mL with sterile distilled water, pipetted 0.5 mL onto 9 agar plates, and incubated at 28 ℃ for 24 h to terminate incubation. Cutting the agar blocks in the whole plate into strips, taking out the strips, dyeing the strips by using a cotton blue dye solution, observing and counting the chlamydospore germination number and non-germination number of the whole plate under an optical lens, and thus obtaining the total number. Wherein, the judgment standard of spore germination is that the spore can germinate when the length of hypha grown from the germination tube is larger than the radius of the spore.
Meanwhile, the spore germination rate is measured by taking the same amount of dry spore powder to serve as a control group, and the method is as follows:
furthermore, the sample capacity is predicted according to the following formula:
where p is the expected frequency of an event and L is the allowable error
When the germination rate of chlamydospores is 8% -30% and the sample capacity n is 736-2100, 95% of reliability reaches the accuracy with the allowable error of 2%. To reduce errors, the total number of chlamydospores counted per experiment was therefore greater than 2000. Finally, statistical data were plotted using GraphPad Prism 6.0.
The results of the chlamydospore germination rate measurements in lick blocks are shown in FIG. 2.
As can be seen from FIG. 2, the different formulations had inconsistent effects on the germination rates of chlamydospores in lick blocks, indicating that the germination rates of spores in the control group were 35.04%, while those in examples 1, 2 and 3 were 27.32%, 32.74% and 38.65%, respectively. Thus, example 3 is a preferred example, more suitable for germination of spores in lick blocks.
Example 5 insecticidal Effect assay of chlamydospore lick blocks on infective larvae in sheep feces
8 small tailed Han sheep (3-4 months old) were anthelmintically combined with albendazole (15 mg/kg body weight) and levamisole (7.5 mg/kg body weight) to repel mixed parasitic worms (including cestodes) infected in the test animals. After expelling parasites for 7 days, the stored animal dung is inspected for egg by saturated saline floating method and no other miscellaneous nematode infection is proved, and then the stored animal dung is inspected under an inverted microscopeH. contortusL3 viability, after passing the examination, L3 was diluted with distilled water, and the whole volume (50. mu.L) was placed on a slide glass and counted under a microscope, and 3 times of continuous sampling were performed to obtain the average.
The quantity of L3 contained in each milliliter of the original larval liquid is calculated according to the counting result so as to inoculate the sheep. After L321 d was artificially inoculated, the feces were examined for EPG by the modified Macmarster method, and it was examined whether EPG was 500 or more. The sheep manure worm eggs are counted, and each gram of manure has similar worm egg number (1870 +/-912).
The 8 sheep infected with similar ova were treated with 8 licking blocks (500 g/block) containing no fungal spores prior to the official test. The lick block formulation was the same as example 3 except that no spore powder was added.
The test sheep were allowed to eat for 5 days, and after 5 days, uneaten licking blocks were collected and weighed, and the average daily consumption of the licking blocks by each test sheep was calculated, which indicated that the amount of licking blocks consumed in 5 days was 2240 g in 8 test sheep, and the average daily consumption of licking blocks was 56 g in each test sheep. Feeding tests show that the test animals show good feeding performance on the lick block.
After feeding is finished, dividing 8 test sheep into 2 groups (namely a test group and a control group, wherein each group comprises 4 sheep) at random, and separately feeding;
the test group was administered with 4 sheep containing the composition prepared in example 3D. flagransThe block is continuously thrown for 7 days, the feces of the test sheep are collected once a day after the 2 nd day, the feces of each sheep are divided into 3 parts, 10 g of each part is weighed, the parts are placed in a culture dish of 4.5 cm and are crushed by a pair of tweezers and are stirred evenly by toothpicks, a proper amount of distilled water and sawdust after autoclaving are added to adjust the humidity and ventilate, and then the parts are placed in a large plate of 7.5 cm in diameter, wherein 10 mL of distilled water is added, and the plates of the test group are arranged in 12 parallels.
In addition, the control group of sheep was fed with the same number of licking blocks without fungus and allowed to feed freely, and feces culture, larva isolation and counting were performed in the same manner as in the test group starting at the 2 nd feeding, and 12 replicates were also set. The average number of L3 obtained in each group was calculated by counting larvae as described by Cai et al (2017) and the formula for the kill rate was as follows:
the average of L3 in the control and test groups was calculated using SPSS (Version 23.0) software and the significance of the difference was judged at a 5% probability level by analysis of variance.
The results of the in vivo efficacy test conducted in the laboratory on the lick block are shown in table 1;
as can be seen from Table 1, the average number of L3 in feces of the test group was significantly lower than that of the control group at 1-7 d of the test period: (p< 0.05). The average number of L3 in the feces of the two groups is still significantly different after the licking block feeding is stopped for 1-3 d. In the 4 th day of stopping feeding lick block, the insecticidal rate is reduced to 57.85%, and the difference is significant (p< 0.05). The results show that the chlamydospore licking block of the nematophagous fungus has obvious insecticidal rate to L3 in excrement after being fed to test animals, and the chlamydospore licking block of the nematophagous fungus has obvious effect on biological control of the gastrointestinal nematode diseases of cattle and sheep.
Example 6 evaluation test of storage stability of lick Block
Test for determination of storage stability of lick blocks under different storage conditions:
the following six different storage conditions were selected: the licking block is prepared by the method of example 3, and the licking block samples are respectively stored for 1-7 months in indoor and outdoor conditions at-20 ℃, 4 ℃, outdoor sun-shading and simulated warehouses. Control group: the lick block formulation was the same as example 3 except that no spore powder was added.
(II) detecting the insecticidal effect under different storage conditions:
the licking block samples are respectively placed indoors and outdoors at the temperature of minus 20 ℃, 4 ℃, outdoors and in a sun-shading and simulated warehouse, and are sampled once every month to detect the insecticidal rate. Feces culture was performed according to the method used in example 5, and the third-stage larvae were separated by the Bellman method, counted, and the insecticidal ratio was calculated.
(III) data processing: the experimental data were analyzed for variance using SPSS software (22.0), with Duncan (D) method being chosen for pairwise comparisons.
(IV) shelf life measurement results
The in vitro insecticidal effect of the test licking block stored under the 6 different conditions for 1-7 months is shown in figure 3As can be seen in FIG. 3, the amount of L3 in feces was significantly lower in all test groups (lick blocks containing fungi) than in the control group (seep< 0.05). The licking block is stored for 1-7 months under indoor conditions, and the in-vitro insecticidal rate of L3 in feces is 98.51% -99.75%; the licking block is stored for 1-7 months under outdoor conditions, and the in-vitro insecticidal rate of L3 in feces is 37.43% -99.79%; the licking block is stored for 1-7 months under the outdoor sun-shading condition, and the in-vitro insecticidal rate of L3 in excrement is 89.48% -99.27%; the licking block is stored for 1-7 months under the condition of a simulated warehouse, and the in-vitro insecticidal rate of L3 in excrement is 86.43% -99.45%; the licking block is stored for 1-7 months at 4 ℃, and the in-vitro insecticidal rate of L3 in feces is 96.06% -99.87%; the licking block is stored for 1-7 months at the temperature of-20 ℃, and the in-vitro insecticidal rate of L3 in excrement is 94.53% -99.47%.
In conclusion, the licking block preparation is stored indoors and in a warehouse at the temperature of 4 ℃ to-20 ℃, and the insecticidal rate of more than 90 percent is still kept when the licking block preparation is stored for 7 months, so that the storage condition in common daily use can be met, large-scale instruments and equipment are not needed, the electric power is saved, the cost is reduced, and the storage and the transportation in future popularization are facilitated.
Except for special description, the proportions are weight ratios, and the percentage contents are mass percentage contents.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A preparation method of a chlamydospore licking block of nematophagous fungi is characterized in that: the licking block component adopted by the preparation method comprises the following components in parts by weight: 5-10 parts of urea, 15-20 parts of molasses, 10-25 parts of salt, 5-10 parts of sodium bentonite, 0-2 parts of carboxymethyl cellulose and monocalcium phosphate0 to 5 parts of vitamin complex, 1 to 1.5 parts of composite vitamin, 20 to 30 parts of bran, 1 to 2 parts of peanut oil,D. flagrans1-2 parts of chlamydospore powder, wherein the licking block contains 0.5-2 × 104Per g isD. flagransChlamydospores.
2. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the preparation method comprises the following steps: the adopted strain is flagellum type ArthrobotrysArthrobotrysflagransIsolate SDH035 with preservation number CGMCC NO. 11210.
3. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the preparation method comprises the following steps: comprises thatD. flagransPreparation of chlamydospore powder: flagellate type arthrobotrys of nematode-trapping fungiD. flagransAnd carrying out batch culture on the SDH035 isolate on a corn-wheat combined grain culture medium, wherein the culture temperature is 25-30 ℃, the relative humidity is 80-100%, and the culture time is 15-25 d, and then drying the grain containing spores until the moisture content in the grain is below 15%.
4. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 3, which is characterized in that: the above-mentionedD. flagransPreparation of chlamydospore powder, wherein the spore content of the prepared chlamydospore powder is 1-2 × 106Per gram.
5. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the preparation method comprises the following steps: the preparation method comprises the following steps: firstly, 30% of bran powder by mass is weighed, then the spore powder is added into the bran powder, and the two are fully and uniformly mixed; the bran powder containing the spore powder is then thoroughly mixed with the remaining bran powder and the remaining components of the formulation.
6. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the licking block comprises the following components in parts by weight: 25 parts of urea, 75 parts of molasses, 105 parts of NaCl, 25 parts of sodium bentonite, 15 parts of carboxymethyl cellulose, 10 parts of monocalcium phosphate, 5 parts of composite vitamin, 225 parts of bran powder, 5 parts of spore powder and 10 parts of edible peanut oil.
7. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the lick block prepared: the spore germination rate reaches over 27.32-38.65%.
8. The application of the chlamydospore lick block of the nematophagous fungus is characterized in that: the licking block is used for feeding cattle and sheep, and the insecticidal rate of gastrointestinal nematodes reaches more than 83%.
Priority Applications (1)
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| CN105362301A (en) * | 2015-11-11 | 2016-03-02 | 西北民族大学 | Nematophagous fungus granular preparation as well as preparation method and application thereof |
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| CN105961860A (en) * | 2016-05-23 | 2016-09-28 | 四川农业大学 | Anti-coccidium block for sheep and preparation method of anti-coccidium block |
| CN106173209A (en) * | 2016-07-21 | 2016-12-07 | 蔡葵蒸 | A kind of nematode-destroying fungus powderous preparations and application thereof |
| CN106417916A (en) * | 2016-09-29 | 2017-02-22 | 张元生 | Licking blocks for ruminants to lick and preparation method thereof |
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| CN101971936A (en) * | 2010-11-13 | 2011-02-16 | 兰州大学 | Special compound additive lick brick for Tibetan sheep |
| CN103783284A (en) * | 2013-06-07 | 2014-05-14 | 昆明赛德饲料有限公司 | Multi-nutrient block for promoting fattening of cattle and sheep and breeding stock health |
| CN105362301A (en) * | 2015-11-11 | 2016-03-02 | 西北民族大学 | Nematophagous fungus granular preparation as well as preparation method and application thereof |
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| CN106417916A (en) * | 2016-09-29 | 2017-02-22 | 张元生 | Licking blocks for ruminants to lick and preparation method thereof |
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